The type 1-interferon (IFN) response is the first line of defense against viral infections. Hundreds of proteins are induced in IFN-stimulated cells, and these proteins mediate a wide spectrum of anti-viral effects. ISG15 was one of the first IFN-induced proteins to be identified and the first ubiquitin-like protein (Ubl) to be discovered, however its biochemical function and the basis of its antiviral activities have remained largely uncharacterized for many years. ISG15 has anti-viral activity against a wide range of human viruses, including influenza, retroviruses, sindbis, ebola, and the ability of ISG15 to be conjugated to other proteins is likely to be essential for its anti-viral activity in all cases. ISG15 is conjugated to hundreds of cellular proteins in IFN-stimulated cells, and a single IFN-induced ligase, Herc5, mediates conjugation to nearly all of these target proteins. Based on extensive preliminary data, we hypothesize that Herc5 is ribosome-associated and ISGylates proteins in a co-translational manner, with little target protein specificity. We further hypothesize that in the context of an interferon response, newly translated viral proteins, rather than cellular proteins are the primary targets of this system, and that ISGylation is an attempt to inactivate the function of viral proteins. The aims of this proposal will test both of these hypotheses. The interferon response is a mutli-faceted defensive shield that protects cells against a wide range of infectious agents, yet only a small number of IFN-induced proteins have been characterized in molecular detail. In addition, interferon is used therapeutically in treating viral infections (e.g., hepatitis B and C and papillomaviruses), as well as certain types of cancers and multiple sclerosis, yet it is not known which IFN-induced proteins mediate useful therapeutic effects and which mediate the notorious side effects of these therapies. A further understanding of the mechanism and function of ISG15 conjugation will present opportunities for either up- or down-modulating ISGylation activity in these clinical settings. PUBLIC HEALTH RELEVANCE: ISG15 conjugation is now recognized to be an important aspect of the innate immune response against a wide range of human viral infections, and interferon therapies are approved for many disease states. It is essential to characterize the many facets of the interferon response system, including the ISG15 pathway, in order to improve and modulate anti-viral and interferon therapies.